The ATP energy-transducing system (ATPase complex) in membranes of Escherichia coli functions as a transducer of the energy in the phosphate-ester bond of ATP to an electrochemical potential across the membrane, or vice versa. To do this, the complex couples the translocation of protons across the membrane to the hydrolysis of ATP. This energy-transducing system is required for oxidative phosphorylation and can energize the membrane to drive active transport. The object of the proposed research is to: (1) Verify that the proton translocase is a component intrinsic to the ATPase complex; (2) Identify the components of the proton-translocase; (3) Reconstitute the proton-translocase activity from purified components in the artificial membrane. The strategy relies on the susceptibility of the proton-translocase to inhibition by dicyclohexylcarbodiimide. This property will be used as the identifying chemical feature in isolation of the translocase. I have purified the membrane protein which reacts with dicyclohexylcarbodiimide to cause inhibition of the proton-translocase. The protein, which may itself be the translocase-component of the complex, will be characterized chemically and tested for proton-translocation after reconstitution with phospholipid in an artificial membrane. The ATPase complex, when extracted with mold detergents, contains 5 - 7 subunits of unknown function. The subunits which are involved in proton-translocation will be identified, through isolation of mutants and in vivo and in vitro complementation analyses. These proteins will then be purified and the proton-translocase reconstituted from the isolated components and lipid. It might then be feasible to reconstitute the entire ATP-driven proton-pump.